The liquid can make its way into the pipette and cause contamination to the next sample(s) you use it for or it can just damage the sensitivity of the pipette by getting stuff gunked up inside.
Great tutorial! But, 13:58 you didn't depict the amplification tree (usually the secondary is polyclonal), so there's multiple secondaries binding to the primary to 'blow up' your signal from the HRP reaction. Besides that: *great channel* here, man! Why did you stop though :( Just as I'm discovering all these helpful channels... I even made a Top 10 channel overview on my own channel, and I'm planning on making a second one with 'smaller channels' like this one. All good stuff for Biomedical/Life Science students!
I'm re-acquainting myself with this stuff after years of not being in a wet lab. Thanks for this detail and the overview vid on your channel. Polyclonal secondary antibodies sounds clever and makes a lot of sense.
This video was everything I wanted! I have a vague idea of everything in here and just needed a refresher on the actual practical use of everything. Thank you!
you prolly dont give a damn but does someone know of a way to get back into an Instagram account? I was stupid lost my account password. I would appreciate any tips you can offer me!
@Axton Jax thanks for your reply. I found the site thru google and im trying it out atm. I see it takes quite some time so I will reply here later with my results.
Thanks, great video! How does the metal stirrer help the process? I usually leave the tank without it being stirred the whole time, would love to know how stirring it helps as I may try that! Thanks so much in advance.
Can the primary and secondary antibodies be diluted in the 5% non-fat milk and then each incubated with the membrane? Rather than doing the blocking step as its on whole step?
You mean put everything in at once at the same time to reduce protocol time? Not with the blocking step for sure, but perhaps you can premix primary and secondary at equimolar concentrations, then you are sure that no excess secondary will deplete your primary if you wash afterwards. Then again, you would loose your 'amplification tree' which could results in not seeing a good signal. Usually the secondary antibody is a polyclonal so multiple HRP-conjugated antibodies bind to the Fc-part of your primary, needed to amplify your signal. I would just stay with the sequential protocol. Or maybe I misread your question (just realized I think): yes, your antibodies should be diluted in the same solution as the blocking, but usually it's more diluted than for the blocking. So if you block with 5%, you stain in 1% or 0.5%. Note that some protocols recommend using BSA as blocking and for dilution...
Usually you should to incubate the membrane with bloaking solution during 1 hour. Then you should wash 3 times and incubate with primary Ab diluted in 5% milk PBS overnight. Then you schould wash 3 times and incubate with secondary Ab diluted in 5% milk PBS during 1 hour. Wash 3 times and make a picture.
Very nice! The closest thing we have to lab practice in the pandemic!
Good video but please
14:51 dont hold the pipette upside down with a used tip on it... ;)
Yas omg
... Or use a computer mouse with gloves on.
can you explain why?
The liquid can make its way into the pipette and cause contamination to the next sample(s) you use it for or it can just damage the sensitivity of the pipette by getting stuff gunked up inside.
Better yet, use filter tips
Great tutorial! But,
13:58 you didn't depict the amplification tree (usually the secondary is polyclonal), so there's multiple secondaries binding to the primary to 'blow up' your signal from the HRP reaction.
Besides that: *great channel* here, man! Why did you stop though :(
Just as I'm discovering all these helpful channels... I even made a Top 10 channel overview on my own channel, and I'm planning on making a second one with 'smaller channels' like this one. All good stuff for Biomedical/Life Science students!
I'm re-acquainting myself with this stuff after years of not being in a wet lab. Thanks for this detail and the overview vid on your channel. Polyclonal secondary antibodies sounds clever and makes a lot of sense.
@@lukehebert6207 Cheers, man!
This video was everything I wanted! I have a vague idea of everything in here and just needed a refresher on the actual practical use of everything. Thank you!
Balls🙏🏻
Nice protocol and instruction. Thank you for the video
I'm interested to do this western blot techniques
i have a question : how tween inhibits detection ? what is the reaction mechanism
you prolly dont give a damn but does someone know of a way to get back into an Instagram account?
I was stupid lost my account password. I would appreciate any tips you can offer me!
@Imran Chandler Instablaster ;)
@Axton Jax thanks for your reply. I found the site thru google and im trying it out atm.
I see it takes quite some time so I will reply here later with my results.
@Axton Jax it did the trick and I now got access to my account again. Im so happy:D
Thanks so much, you saved my account !
@Imran Chandler You are welcome xD
Thanks, great video! How does the metal stirrer help the process? I usually leave the tank without it being stirred the whole time, would love to know how stirring it helps as I may try that! Thanks so much in advance.
my mebrane disloved immedietly in the methanol :(
Nice video, how long for blot
ting at 115 volt?
Dont they make you put your hair up in a lab?
Airisweetheart only with caustic or dangerous substances.
Well, personally I always do wether I am asked to or not, because they get in the way and I can't concentrate
so blotting is blotting? I guess I really need to dig deeper...
Ok I'll see what the weather was not in my head is
I wish you have subtitle..
Thank you! Great video, it helps me a lot!
Splendid presentation, thank you.
Rider
Can the primary and secondary antibodies be diluted in the 5% non-fat milk and then each incubated with the membrane? Rather than doing the blocking step as its on whole step?
You mean put everything in at once at the same time to reduce protocol time? Not with the blocking step for sure, but perhaps you can premix primary and secondary at equimolar concentrations, then you are sure that no excess secondary will deplete your primary if you wash afterwards. Then again, you would loose your 'amplification tree' which could results in not seeing a good signal. Usually the secondary antibody is a polyclonal so multiple HRP-conjugated antibodies bind to the Fc-part of your primary, needed to amplify your signal. I would just stay with the sequential protocol.
Or maybe I misread your question (just realized I think): yes, your antibodies should be diluted in the same solution as the blocking, but usually it's more diluted than for the blocking. So if you block with 5%, you stain in 1% or 0.5%. Note that some protocols recommend using BSA as blocking and for dilution...
Usually you should to incubate the membrane with bloaking solution during 1 hour. Then you should wash 3 times and incubate with primary Ab diluted in 5% milk PBS overnight. Then you schould wash 3 times and incubate with secondary Ab diluted in 5% milk PBS during 1 hour. Wash 3 times and make a picture.
Everything clearly explained to more useful to practice this Western Blotting
Thanking you Sir/Mam
Good
Thank you for inviting me to your game! I played and learnt a lot!
it was such an awesome video
Thank you so much!!
Hair down in the lab?
hugely helpful
Thank you
Very useful video. Thank you.
excellent
Lcnd
Thank you.
thank you, brilliant and accurate explanation